1. Field of Invention
The invention relates to nucleic acid multiplexes, and more particularly to methods of creating them as triplexes and quadruplexes, and furthermore employing them in assays to detect specific nucleic acids.
2. Description of Related Art
The ability of two single-stranded nucleic acid molecules of complementary base sequence to bind specifically to each other has provided the basis for both powerful research and powerful diagnostic tools. Less fully explored than such “conventional hybridization” has been the ability of single stranded molecules to bind to double-stranded targets and the ability of double-stranded molecules to bind to double-stranded targets. The ability to bind to double-stranded targets potentially has advantages over conventional hybridization. These could stem in part from the fact that the double-stranded target would not be denatured, allowing “milder” hybridization conditions and providing a target less prone to becoming a totally random coil. They could also stem in part from the fact that the base-pairing mechanisms would be at least partially different than in conventional hybridization, allowing the possibility for more favorable kinetics and a reduction in the amount of probe needed in the hybridization reaction mixture.
Prior work on creating multiplexes have included:                1) The formation of triplexes as part of the homologous recombination process, a process mediated by the bacterial protein RecA and proteins of similar function in other organisms;        2) The creation of 3-stranded structures during in situ hybridization (e.g., U.S. Pat. No. 5,707,801 of Bresser et al.); and        3) 3-stranded or 4-stranded complexes that rely on Hoogstein-type bonding.        
This prior work does not fully exploit the potential for forming multiplexes. The RecA-mediated process requires a protein. The in situ hybridization processes are based on the principle that the double-stranded intracellular target will locally open its double-stranded structures, providing a single-stranded target that will hybridize according to conventional hybridization principles. Complexes reported to rely on Hoogstein-type polymers are limited to structures that are not true heteropolymers. Rather they require that a given strand be a polypurine or polypyrimidine or very close thereto. See, e.g., Floris et al., “Effect of cations on purine-purine-pyrimidine triple helix formation in mixed-valence salt solutions,” 260 Eur. J. Biochem. 801-809 (1999).
As was the case with triplex nucleic acids, the conventional wisdom regarding quadruplex nucleic acids has been that such peculiar structures only exist under relatively extreme conditions for a relatively narrow class of nucleic acids. In particular, Sen et al. (Nature 334:364-366 (1988)) disclosed that guanine-rich oligonucleotides can spontaneously self-assemble into four-stranded helices in vitro. Sen et al. (Biochemistry 31:65-70 (1992)) disclosed that these four-stranded complexes can further associate into superstructures composed of 8, 12, or 16 oligomers.
Marsh et al. (Biochemistry 33:10718-10724 (1994), and Nucleic Acids Research 23:696-700 (1995)) disclosed that some guanine-rich oligonucleotides can also assemble in an offset, parallel alignment, forming long “G-wires”. These higher-order structures are stabilized by G-quartets that consist of four guanosine residues arranged in a plane and held together through Hoogsteen base pairings. According to Sen et al. (Biochemistry 31:65-70 (1992)), at least three contiguous guanines within the oligomer are critical for the formation of these higher order structures.
It has been suggested that four-stranded DNAs play a role in a variety of biological processes, such as inhibition of HIV-1 integrase (Mazumder et al., Biochemistry 35:13762-13771 (1996)), formation of synapsis during meiosis (Sen et al., Nature 334:364-366 (1988)), and telomere maintenance (Williamson et al., Cell 59:871-880 (1989)); Baran et al., Nucleic Acids Research 25:297-303 (1997)).
It has been further suggested that controlling the production of guanine-rich quadruplexes might be the key to controlling such biological processes. For example, U.S. Pat. No. 6,017,709 to Hardin et al. suggests that telomerase activity might be controlled through drugs that inhibit the formation of guanine quartets.
U.S. Pat. No. 5,888,739 to Pitner et al. discloses that G-quartet based quadruplexes can be employed in an assay for detecting nucleic acids. Upon hybridization to a complementary oligonucleotide, the G-quartet structure unfolds or linearizes, thereby increasing the distance between a donor and an acceptor on different parts of the G-quartet structure, resulting in a decrease in their interaction and a detectable change in a signal (e.g., fluorescence) emitted from the structure.
U.S. Pat. No. 5,912,332 to Agrawal et al. discloses a method for the purification of synthetic oligonucleotides, wherein the synthetic oligonucleotides hybridize specifically with a desired, full-length oligonucleotide and concomitantly form a multimer aggregate, such as quadruplex DNA. The multimer aggregate containing the oligonucleotide to be purified is then isolated using size-exclusion techniques.
Despite the foregoing developments, the full potential of quadruplex nucleic acid has neither been fully appreciated nor fully exploited.
Related to the problem of performing hybridization-type experiments with double-stranded targets is the means of detecting them. Fluorescent dyes have been used to detect and quantitate nucleic acids for decades. In their most basic form, fluorescent intensity-based assays have typically comprised contacting a target with a fluorophore-containing probe, removing any unbound probe from bound probe, and detecting fluorescence in the washed sample. Homogeneous assays improve upon such basic assays, in that the former do not require a washing step or the provision of a non-liquid phase support.
For example, U.S. Pat. No. 5,538,848 to Livak et al. and U.S. Pat. No. 4,220,450 to Maggio disclose homogeneous fluorescence-based assays of nucleotide sequences using oligonucleotide probes in solution. However, these patents require the use of a quenching agent in combination with a reporting agent, so as to distinguish between the signals generated by hybridized probes and unhybridized probes. Livak et al. also requires the use of enzymes in its disclosed method. Quenching agents and enzymes add complexity and expense to the methods.
U.S. Pat. No. 5,332,659 to Kidwell discloses a method for detecting nucleotide sequences in solution using probes comprising at least two fluorophore moieties. The fluorophores must be selected to electronically interact with each other when close enough to vary the wavelength dependence of their spectra. Unhybridized probes are much more flexible than probes hybridized to the target sequence, and consequently the two fluorophore moieties on each probe are more likely to be close to each other when the probe is unhybridized than when the probe is hybridized. Thus, a change in emission wavelength correlated with free probe can be monitored as an indication of the amount of free probe in the sample.
U.S. Pat. No. 5,846,729 to Wu et al. also discloses homogeneous fluorescence-based assays for detecting nucleic acid.
In addition to the aforementioned developments which detect fluorescent intensity, some have touted the advantages of fluorescent polarization assays. However, there are significant drawbacks to polarization-based assays. The degree of change in polarization as a function of binding can be unpredictable, and interpretation of data to conform inconsistent data to theoretical expectations can require more effort than is desirable in an analytical method, particularly when the method is to be automated. There are as well constraints arising from the molecular weight of the molecules whose motion is being evaluated in a fluorescent polarization assay.
The present inventions will be seen, in various important embodiments to take advantage of the properties of fluorescent molecules for purposes of detecting triplexes and quadruplexes.
All references cited herein are incorporated herein by reference in their entireties.